Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι.

Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PB1-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that selectively inhibit hepatic aPKC may be useful treatments.

The critical role of atypical protein kinase C in activating hepatic SREBP-1c and NFkappaB in obesity.

Obesity is frequently associated with systemic insulin resistance, glucose intolerance, and hyperlipidemia. Impaired insulin action in muscle and paradoxical diet/insulin-dependent overproduction of hepatic lipids are important components of obesity, but their pathogenesis and inter-relationships between muscle and liver are uncertain. We studied two murine obesity models, moderate high-fat-feeding and heterozygous muscle-specific PKC-lambda knockout, in both of which insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but conserved in liver. In both models, activation of hepatic sterol receptor element binding protein-1c (SREBP-1c) and NFkappaB (nuclear factor-kappa B), major regulators of hepatic lipid synthesis and systemic insulin resistance, was chronically increased in the fed state. In support of a critical mediatory role of aPKC, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP-1c and NFkappaB, and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Moreover, in high-fat-fed mice, impaired insulin signaling to IRS-1-dependent phosphatidylinositol 3-kinase, PKB/Akt and aPKC in muscle and hyperinsulinemia were largely reversed. In obesity, conserved hepatic aPKC-dependent activation of SREBP-1c and NFkappaB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic insulin resistance. Accordingly, hepatic aPKC is a potential target for treating obesity-associated abnormalities.

Muscle-specific knockout of PKC-lambda impairs glucose transport and induces metabolic and diabetic syndromes.

Obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM) are major global health problems. Insulin resistance is frequently present in these disorders, but the causes and effects of such resistance are unknown. Here, we generated mice with muscle-specific knockout of the major murine atypical PKC (aPKC), PKC-lambda, a postulated mediator for insulin-stimulated glucose transport. Glucose transport and translocation of glucose transporter 4 (GLUT4) to the plasma membrane were diminished in muscles of both homozygous and heterozygous PKC-lambda knockout mice and were accompanied by systemic insulin resistance; impaired glucose tolerance or diabetes; islet beta cell hyperplasia; abdominal adiposity; hepatosteatosis; elevated serum triglycerides, FFAs, and LDL-cholesterol; and diminished HDL-cholesterol. In contrast to the defective activation of muscle aPKC, insulin signaling and actions were intact in muscle, liver, and adipocytes. These findings demonstrate the importance of aPKC in insulin-stimulated glucose transport in muscles of intact mice and show that insulin resistance and resultant hyperinsulinemia owing to a specific defect in muscle aPKC is sufficient to induce abdominal obesity and other lipid abnormalities of the metabolic syndrome and T2DM. These findings are particularly relevant because humans who have obesity, impaired glucose tolerance, and T2DM reportedly have defective activation and/or diminished levels of muscle aPKC.

Fusion of antigen to Fas-ligand in a DNA vaccine enhances immunogenicity.

DNA vaccines have considerable potential for the prophylaxis and therapy of a range of diseases, but their potential has not been realised largely due to poor immunogenicity. Fas ligand is a pro-apoptotic molecule, able to induce death of Fas expressing cells. We describe the construction of a DNA vaccine encoding a chimeric fusion between Fas ligand and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T helper 2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein.

Enhancement of immune responses to an HIV gp120 DNA vaccine by fusion to TNF alpha cDNA.

DNA vaccines have considerable potential for disease prophylaxis and therapy, but are generally poorly immunogenic. A number of means of enhancing immunogenicity have been assessed, including the co-expression of cytokines, the use of heterologous prime-boost regimes, and the addition of more conventional adjuvants. In this study we have assessed the effects on gp120 DNA immunogenicity of in-frame fusion of tumor necrosis factor alpha DNA to DNA encoding a large fragment of HIV gp120. The studies were performed using a DNA prime, protein boost regime and a heterologous boosting protein. Fusion of TNFalpha DNA enhanced Th1 related immune responses against both the priming and the boosting gp120. In-frame fusion of interferon gamma-encoding DNA at the 5' end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity.

An interferon gamma-gp120 fusion delivered as a DNA vaccine induces enhanced priming.

Nucleic acid vaccination has many potential advantages over traditional methods, but suffers from the fact that DNA vaccines tend to be relatively poorly immunogenic. Attempts to enhance DNA vaccine immunogenicity have included the addition of cytokine-encoding plasmids into the formulation, as well as the use of heterologous prime-boost regimes and the addition of conventional adjuvants, such as alum. We have previously shown that interferon gamma fusions have enhanced immunogenicity as recombinant protein vaccines. We have assessed here the immunogenicity of an interferon gamma-gp120 fusion delivered as a DNA vaccine, in the context of a prime-boost strategy and in the presence of absence of aluminium phosphate. Fusion of gp120 DNA to interferon gamma-encoding DNA resulted in strongly enhanced priming, especially of Th1 responses, including IgG2a responses to a protein boost.